Chemokines and their receptors control cell migration in development, in immune system homeostasis, and in inflammatory responses to physiological insults. Chemokine receptors CXCR4 and ACKR3 also play critical roles in disease, particularly cancer where they work as a trio with their mutual chemokine ligand, CXCL12. CXCR4, a canonical G-protein coupled receptor (GPCR) drives tumor metastasis by virtue of its ability to promote cell migration. In contrast, ACKR3 is an atypical receptor that does not couple to G proteins; instead it signals via ?-arrestin and indirectly facilitates metastasis by biased ?-arrestin mediated signaling. As such, CXCR4 and ACKR3 represent promising targets for the development of novel anti-cancer therapeutics. Despite their importance in cancer, the structural basis of ligand recognition and activation of these two receptors by CXCL12 remains a mystery. In fact, the question of recognition of any protein ligand by a GPCR was unanswered until recently when the applicants solved the first structure of a CXCR4:chemokine complex. This was a major breakthrough that poised the applicants well for studying the molecular basis of the interaction of CXCR4 and ACKR3 with CXCL12, and how they function as canonical G protein-coupled and ?- arrestin biased receptors. The long term goal of the applicants' research is to obtain atomic resolution understanding of chemokine receptor interactions with their protein ligands and intracellular partners, thus enabling rational design of highly efficient therapeutics targeting these chemokine receptors. The objective of the present proposal is to understand the structural basis of how CXCL12 is recognized by two functionally distinct receptors and how ligand binding is translated into distinct functional responses. The proposal consists of three complementary Specific Aims. In Aim 1, the structural basis of CXCR4 and ACKR3 interaction with CXCL12 and small molecules will be determined using X-ray crystallography and experiment-guided molecular modeling. In Aim 2, the pharmacological peculiarities of CXCR4 and ACKR3 activation and signaling will be deconvoluted via model-guided binding, kinetic, and functional experiments. In Aim 3, the complex dynamics and the conformational changes that the canonical GPCR CXCR4 and the atypical biased receptor ACKR3 undergo in response to ligand binding will be investigated using EPR. Our comprehensive interdisciplinary approach involves crystallography, molecular modeling, a wide range of experiments for studies of receptor binding and signaling, and EPR characterization of receptor conformational changes and dynamics. The overall project is innovative because of its structural hypotheses, its synergistic methodologies, and because this is the first time a complex polypharmacological system of two membrane receptors (one of which is atypical) and several shared ligands of varying specificity and pharmacology is studied from a structural perspective and in the context of full-length receptors. The research is significant, because it is expected to vertically advance both chemokine receptor biology and the development of novel cancer therapeutics.